F1F0-type are an important group of membrane-bound multisubunit enzymes involved in using ATP to generate a proton gradient, which can be used for substrate or ion transport. The primary role of these enzymes in mitochondria or chloroplasts in eukaroyte cells is the synthesis of the cellular fuel, ATP. Emerging evidence is that the F1F0 ATPase is a rotary motor, the smallest yet known. Understanding how this enzyme works will aid in identifying and characterizing other cellular motors. Ongoing studies in this laboratory are aimed at defining the molecular details of how the cooperative functioning of three catalytic sites in ATP hydrolysis or ATP synthesis is coupled to proton translocation through a proton pore. This appears to involve a rotor including the gamma-epsilon and 12 c subunits of the enzyme moving against a stator of the alpha3beta3deltab2 and a subunits in the F1F0 from escherichia coli. Crystallographic and electron microscopy approaches are being used to examine the structure of the enzyme at different positions in the rotation cycle. In humans, two of the subunits of the F1F0, subunits 6 and A6L, are encoded on mitochondrial (mt) DNA. Pathogenic mutants of mtDNA are being reported with increased frequency, including ones in subunits 6. Human F1F0 has been isolated from fibroblast cell lines for the first time in this laboratory. Studies are planned to examine the enzymatic properties and the assembly of F1F0 in cells of patients harboring subunit 6 mutations.